P1405: IN-DEPTH GENOME INTEGRITY EVALUATION AND GMP-COMPLIANT MANUFACTURING OF HDR GENE EDITED CD4+ T CELLS FOR THE TREATMENT OF HYPER IGM 1
Authors: Daniele Canarutto, Claudia Asperti, Valentina Vavassori, Simona Porcellini, Elisabetta Rovelli, Marianna Paulis, Samuele Ferrari, Tiziana Plati, Lucia Sergi Sergi, Anna Villa, Marina Radrizzani, Luigi Naldini
Journal: HemaSphere (2023)

Topic: 25. Gene therapy, cellular immunotherapy and vaccination - Clinical Background: We have been developing a treatment for Hyper IgM 1, a combined immunodeficiency caused by mutations in the X-linked CD40LG gene, by homology driven recombination (HDR) gene editing using Cas9 and a viral donor template. Anticipating clinical translation, we have designed a suitable manufacturing process. Furthermore, as there is no consensus on the characterization of gene editing products, we developed suitable assays to assess genomic outcomes following editing. Aims: Large scale GMP manufacturing of edited CD4+ T cells Characterize the genome integrity of CD4+ T cells gene edited with Cas9 and a corrective donor template Methods: CD4+ T cells were edited with Cas9 and adenovirus associated virus serotype 6 (AAV6) or integrase defective lentivirus vector (IDLV) carrying a corrective partial cDNA cassette and a functionally linked NGFR reporter, that could be exploited for the enrichment of edited cells. Development of a suitable manufacturing process was performed by systematic comparison of raw materials. On-target large deletions were detected with custom ddPCR assays overlapping neighboring genes. High coverage (1400X) optical mapping was analyzed with the annotated rare variant analysis, comparing each sample with its untreated counterpart. CD40L expression and binding to CD40 were assessed by flow cytometry. CD40 signal transduction was assessed by co-culturing cells with CD40L Hek-blue cells. Results: Starting from a research grade protocol, we developed a 2 week-long Good Manufacturing Practice (GMP)-compliant large scale drug manufacturing process using CliniMACS Prodigy and Grex 100M, that allows for long range editing, enrichment and expansion of edited cells, delivering a highly pure product with preserved T-stem cell central memory and central memory phenotype, and documented potency. We found large deletions occurring on-target upon editing with AAV6 donor that were counter-selected in culture. To overcome the limits of conventional karyotype, we validated high resolution optical mapping on a custom edited cell line; we were able to detect a 6.9 kb insertion at 2.5% variant allele frequency (VAF) and a larger 296 kb rearrangement at 10% VAF. We then analyzed IDLV-edited CD4+ T cells from 3 donors at the end of the manufacturing process, and found large on-site integrations (Figure 1A-Bs), with a VAF of up to 21%. These integration events increased in frequency with the fraction of edited cells, were compatible with template concatemers, and did not appear to compromise functionality of edited cells, as assessed by expression of CD40LG, surface binding to CD40 and downstream signal transduction. No deletions, translocations or other off-target related recurring events were detected.Figure 1: A) optical genome mapping of gene edited CD4 T-cells, after enrichment, from three donors. Insertions (green dots) can be seen in the CD40LG locus of donor #2 and #3 (panel B). The green bar represents the reference genome assembly; blue bars indicate sample-derived assemblies. Trapezius indicates the insertion event. Vertical blue and yellow lines overlapping assemblies correspond to restriction sites present in the reference assembly (blue) or not (yellow). Summary/Conclusion We developed a a large scale GMP process for long range editing of edited CD4 T-cells suitable for clinical translation. On-target large deletions and integrations can be relatively frequent and should be anticipated when developing similar gene editing strategies. High coverage optical mapping allows for unbiased genome wide assessment of genome integrity in these settings. Keywords: Gene therapy, Immunodeficiency, Genomic instability, Genomics